Redox-Regulated Adaptation of Streptococcus oligofermentans to Hydrogen Peroxide Stress

Abstract

Preexposure to a low concentration of H2O2 significantly increases the survivability of catalase-negative streptococci in the presence of a higher concentration of H2O2. However, the mechanisms of this adaptation remain unknown. Here, using a redox proteomics assay, we identified 57 and 35 cysteine-oxidized proteins in Streptococcus oligofermentans bacteria that were anaerobically cultured and then pulsed with 40 μM H2O2 and that were statically grown in a 40-ml culture, respectively. The oxidized proteins included the peroxide-responsive repressor PerR, the manganese uptake repressor MntR, thioredoxin system proteins Trx and Tpx, and most glycolytic proteins. Cysteine oxidations of these proteins were verified through redox Western blotting, immunoprecipitation, and liquid chromatography-tandem mass spectrometry assays. In particular, Zn2+-coordinated Cys139 and Cys142 mutations eliminated the H2O2 oxidation of PerR, and inductively coupled plasma mass spectrometry detected significantly decreased amounts of Zn2+ in H2O2-treated PerR, demonstrating that cysteine oxidation results in Zn2+ loss. An electrophoretic mobility shift assay (EMSA) determined that the DNA binding of Mn2+-bound PerR protein (PerR:Zn,Mn) was abolished by H2O2 treatment but was restored by dithiothreitol reduction, verifying that H2O2 inactivates streptococcal PerR:Zn,Mn through cysteine oxidation, analogous to the findings for MntR. Quantitative PCR and EMSA demonstrated that tpx, mntA, mntR, and dpr belonged to the PerR regulons but that only dpr was directly regulated by PerR; mntA was also controlled by MntR. Deletion of mntR significantly reduced the low-H2O2-concentration-induced adaptation of S. oligofermentans to a higher H2O2 concentration, while the absence of PerR completely abolished the self-protection. Therefore, a low H2O2 concentration resulted in the cysteine-reversible oxidations of PerR and MntR to derepress their regulons, which function in cellular metal and redox homeostasis and which endow streptococci with the antioxidative capability. This work reveals a novel Cys redox-based H2O2 defense strategy employed by catalase-negative streptococci in Mn2+-rich cellular environments. IMPORTANCE The catalase-negative streptococci produce as well as tolerate high levels of H2O2. This work reports the molecular mechanisms of low-H2O2-concentration-induced adaptation to higher H2O2 stress in a Streptococcus species, in which the peroxide-responsive repressor PerR and its redox regulons play the major role. Distinct from the Bacillus subtilis PerR, which is inactivated by H2O2 through histidine oxidation by the Fe2+-triggered Fenton reaction, the streptococcal PerR is inactivated by H2O2 oxidation of the structural Zn2+ binding cysteine residues and thus derepresses the expression of genes defending against oxidative stress. The reversible cysteine oxidation could provide flexibility for PerR regulation in streptococci, and the mechanism might be widely used by lactic acid bacteria, including pathogenic streptococci, containing high levels of cellular manganese, in coping with oxidative stress. The adaptation mechanism could also be applied in oral hygiene by facilitating the fitness and adaptability of the oral commensal streptococci to suppress the pathogens.

Environment-Specific Probiotic Supernatants Modify the Metabolic Activity and Survival of Streptococcus mutans in vitro

“…Note that we cannot fully exclude the observed effects to be associated with other, non-measured probiotic products present in the supernatant, but given the consistency and plausibility of the measured presence of H 2 O 2 and reuterin and the observed effects on S. mutans, the outlined pathway of how probiotics impact on S. mutans seems likely. H 2 O 2 is the major antibacterial substance produced by S. oligofermentans (Zhang et al, 2010;Tong et al, 2020), as confirmed by our study. S. oligofermentans possesses three H 2 O 2 -forming enzyme: lactate oxidase (Lox), that catalyzes L-lactate and oxygen to produce H 2 O 2 and pyruvate; pyruvate oxidase (Pox), that generates H 2 O 2 by oxidizing pyruvate to acetate via acetyl coenzyme; L-amino acid oxidase, that catalyzes the production of H 2 O 2 from amino acids and peptone (Tong et al, 2008;.…”

“…H 2 O 2 is the major antibacterial substance produced by S. oligofermentans ( Zhang et al, 2010 ; Tong et al, 2020 ), as confirmed by our study. S. oligofermentans possesses three H 2 O 2 -forming enzyme: lactate oxidase (Lox), that catalyzes L-lactate and oxygen to produce H 2 O 2 and pyruvate; pyruvate oxidase (Pox), that generates H 2 O 2 by oxidizing pyruvate to acetate via acetyl coenzyme; L-amino acid oxidase, that catalyzes the production of H 2 O 2 from amino acids and peptone ( Tong et al, 2008 ; Liu L. et al, 2012 ).…”

A Novel Aquaporin Subfamily Imports Oxygen and Contributes to Pneumococcal Virulence by Controlling the Production and Release of Virulence Factors

“…5A to D ), we hypothesize that the mechanistic basis of Pn-AqpC in pneumococcal pathogenicity lies in its control of pneumococcal H 2 O 2 production and Ply release. Compared with other pathogenic bacteria, streptococci are highly capable of resisting oxidative stress via endogenous H 2 O 2 -induced resistance to higher levels of exogenous H 2 O 2 ( 6 , 39 ), and this unique characteristic enables them to defend against the innate immune system of the infected host ( 9 ). H 2 O 2 also contributes to pneumococcal virulence by damaging alveolar epithelial cell DNA and suppressing host innate immune systems ( 4 , 5 ).…”

RNase Z Oxidative Degradation Impedes tRNA Maturation and is Involved in Streptococcal Translation Regulation in Response to Oxidative Stress

“…Streptococci are catalase-negative facultative anaerobic bacteria and are well known for producing and tolerating high levels of H 2 O 2 ( 21 – 24 ). Similar to E. coli and B. subtilis , streptococci are capable of regulating antioxidant genes in response to H 2 O 2 ( 25 – 28 ). For example, Streptococcus pneumoniae , a human opportunistic pathogen, employs the response regulator RR14 to sense H 2 O 2 and derepress the expression of antioxidant genes ( 27 ), while Streptococcus oligofermentans , an oral commensal bacterium, utilizes the peroxide-responsive regulator PerR cysteine oxidative inactivation to derepress the expression of oxidative stress defense genes ( 28 , 29 ).…”

“…Similar to E. coli and B. subtilis , streptococci are capable of regulating antioxidant genes in response to H 2 O 2 ( 25 – 28 ). For example, Streptococcus pneumoniae , a human opportunistic pathogen, employs the response regulator RR14 to sense H 2 O 2 and derepress the expression of antioxidant genes ( 27 ), while Streptococcus oligofermentans , an oral commensal bacterium, utilizes the peroxide-responsive regulator PerR cysteine oxidative inactivation to derepress the expression of oxidative stress defense genes ( 28 , 29 ). Previously, we found that S. oligofermentans would pause growth for a period of time when encountering H 2 O 2 ( 28 ), implying that translation regulatory mechanisms could be involved in adaptation to oxidative stress.…”